2. Introduction: Protein Structure
Protein primary
structure arises from
the protein’s specific
amino-acid sequence.
Amino acid sequence
is dictated by the
central dogma: DNA
→ mRNA→ Codon →
Polypeptide (amino
acid sequence)
3. Relating Structure with Function
Depending on the chemical
properties of the amino acids in the
primary structure, the protein folds
into a specific secondary structure
containing α helices and β pleated
sheets.
The protein’s actual 3-D
conformation is dictated by the
specific intermolecular interactions
between amino acids in these α
helices and β pleated sheets.
Changes in tertiary structure can
alter protein function by altering the
amino acid interactions and thus
changing the overall shape of the
protein.
4. Mutations
Genetic mutations occur when errors occur in DNA replication or
RNA transcription. These errors may change the amino acid
sequence and alter protein conformation at the location of the
mutation.
Protein function may be compromised, as illustrated in this figure
below.
5. Human NADPH-Cytochrome P450 Reductase
• Human Cytochrome P450 Oxidoreductase (POR, shown
below), is an enzyme in the liver and other organs that
transfers electrons to the following cofactors, which are
reduced as they accept electrons:
NADPH →→ FAD →→ FMN →→ Heme (Fe)
• The heme group
contains a central
Iron atom which
has high affinity
for electrons.
6. Functions of POR
Various other Cytochrome
P450s found throughout
the body are dependent
upon POR’s electron
transfer to them.
These Cytochrome P450s
are responsible for
carrying out important
processes such as drug
metabolism and steroid
synthesis, as shown in the
figure to the right.
7. POR Mutations: Antley-Bixler Syndrome
Certain mutations of POR have been identified in human
patients and classified based on the amino acid that has
been altered.
These patients seem to exhibit symptoms of Antley-Bixler
Syndrome, a rare genetic condition associated with a loss
of function
in POR in which
craniofacial
dysmorphism
and/or altered
steroidogenesis
occurs as a result
(Miller, 2004).
Human Skull
8. POR Mutations: Location in POR
The following figure shows the locations of various mutations that
have been identified in POR.
In this diagram, the FMN binding domain is violet, the FAD binding
domain is blue, and the NADPH- binding domain is in green.
A green dot indicates that
the mutation is “silent” and
barely impacts POR function,
while a red dot indicates that
POR activity is less than 25%
of the wild type POR activity.
Note the approximate location
of the highlighted S102P and
R550Q mutations whose
structure-function
relationships are the subject
of this study. S102P R550Q
9. Impaired POR Function in Mutants
Research in Dr. Masters’ Lab indicates that binding affinity
for FAD, a cofactor in electron transfer, is diminished in both
the V492E and R457H POR human mutations. The human
patients with these defects have impaired steroidogenesis
and craniofacial defects.
In these two locations,
the mutation causes
changes in enzyme
structure at the FAD
binding site, resulting
in enzyme instability
and impairment of POR
activity compared with
the wild type protein as
shown in this electron
density diagram.
10. S102P and R550Q Mutations
The S102P (Serine to Proline
substitution) and R550Q (Arginine
to Glutamine) mutations were
originally found in human subjects
from the Czech Republic.
It is unknown whether S102P
and/or R550Q impairs POR
functionality, since these subjects
are anonymous and, thus, any
accompanying illness is not
known.
For S102P, it is suspected that the
proline substitution will disrupt the
helical structure (creating a
“kink”), disrupting the
conformation of the protein in a
crucial location in the molecule.
S102P R550Q
11. Objectives/Hypothesis
Since many mutations in proteins can be innocuous,
we propose to characterize the S102P and R550Q
POR mutants for catalytic function as well as
structural aberrations, compared with wild type POR.
(Previously studied mutants were shown to have
lowered FAD or FMN binding affinities by the
Masters lab, suggesting that riboflavin therapy
could be efficacious.)
It is expected that there will be a difference between
the electron transfer rates of the S102P, R550Q
mutants and the wild type POR when a cytochrome c
reduction assay is performed. These studies are in
progress.
12. Protein Expression
In order to study the effects of the mutation on the native
functionality of POR, a fragment of human DNA, which codes
for POR, is inserted into an E. coli plasmid vector to transfer
the POR DNA into the bacteria to be synthesized into protein.
The E. coli cells are grown with shaking in broth media in 3-
liter flasks.
The protein expressed
by these cells is purified
and collected for enzyme
activity analysis
(e.g., Cytochrome c
reduction assay).
13. Transformation Experiments
• Mutant POR is expressed by
the pET 28a vector E.coli
plasmid, which contains an
antibiotic resistance gene.
• The vector is inserted into E.
coli BL-21 cells, and then the
cells are plated with agar,
which has the antibiotic
corresponding with the
antibiotic resistance gene in
the vector.
• The resulting colonies
appear as white dots on the
agar plates and represent
individual cells.
14. Growth and Induction
• The cells are bumped up to TB media at 37⁰C and grown in
flasks for 24 hours on a shaker.
• Growth is monitored by measuring absorbance of the media
with a spectrophotometer.
• When the cells are
growing fastest at log
phase with an
absorbance of 0.6-0.8,
a chemical called IPTG
is added; this process,
called induction, allows
the cells to start
expressing the protein
of interest.
16. Harvesting
After induction, the cells were spun down in the centrifuge.
The resulting pellet was collected and frozen.
After thawing, the pellet was homogenized with lysate buffer
to release the protein from the cells and centrifuged to rid the
sample of cell debris, and the supernatant was collected for
purification
17. Protein Purification
Nickel Affinity Chromatography is a method of obtaining
purer protein. The proteins are “tagged” by adding
additional histidine residues to the terminal end. The Ni
column captures the histidines specifically and leaves
untagged proteins behind.
18. Cytochrome c Reduction Assay
This diagram shows the electron transfer mechanism
occurring between wild type POR and its associated
Cytochrome P450 (NADPH → FAD → FMN → Heme group).
The Cytochrome c reduction assay measures electron
transfer activity of POR
through the absorbance
changes in Cytochrome
c, which is reduced
and measured at 550 nm.
Alternatively, activity
of the associated
Cytochrome P450
can be measured.
19. Acknowledgements
I would like to thank:
My mentors:
Dr. Satya Panda and Dr. Bettie Sue Masters, as
well as Ms. Karen McCammon and Ms. Katie
Hinchee-Rodriguez for their guidance and
assistance in this project.
My teacher:
Ms. Catherine Gonzalez for teaching the
principles of scientific research.
20. References
Hart S, Wang S, Nakamoto K, Wesselman C, Li Y, Zhong X (2008).
Genetic Polymorphisms in cytochrome P450 oxidoreductase
influence microsomal P450-catalyzed drug metabolism.
Hart S, Zhong X (2008). P450 oxidoreductase: genetic
polymorphisms and implications for drug metabolism and toxicitiy.
Marohnic CC, Panda SP, Martasek P, Masters BS (2006). Diminished
FAD Binding in the Y459H and V492E Antley-Bixler Syndrome
Mutants of Human Cytochrome P450 Reductase. J Biol Chem 281:
35975-35982.
Xia C, Panda SP, Marohnic CC, Martasek P, Masters BS, Kim JP
(2011). Structural basis for human NADPH-cytochrome P450
oxidoreductase deficiency. Proceedings of the National Academy of
Sciences 108: 13486-13491.
21. Picture References
http://www.docstoc.com/docs/22467824/NADPH-Cytochrome-P450-
Oxidoreductase
http://www.google.com/imgres?imgurl=&imgrefurl=http%3A%2F%2F
www.quora.com%2FHow-does-IPTG-induced-gene-expression-work-
at-a-molecular-level
http://www.uky.edu/Pharmacy/ps/porter/CPR.htm
http://www.chempep.com/ChemPep-Generic-Term_Protein.htm
http://www.addgene.org/plasmid_protocols/bacterial_transformation/
http://www.bio-rad.com/en-us/product/protein-stains
All other pictures and charts are either student-generated or used with
mentor permission
